Standard chemical analyses, traditional microscopic methods as well as digital imaging techniques such as confocal scanning laser microscopy, have transformed the structural and functional understanding of biofilms. Investigators, with these techniques have a clearer understanding of biofilm-associated microorganism cell morphology and cellular functions. The heightened awareness of metabolic biochemistry and the events associated with them have led to a better understanding, not only of individual cells and their varying environments, but also collections of cells that form colonies. Further, certain relationships of colonies to each other are under the direct influence of the biofilm in which they reside.
Concurrent with the increased understanding of cellular activity and inter-colony relationships, there has been an awareness developed about the biofilm in which the cells reside. While there has been an increased understanding of the architecture and composition of the biofilm matrix, the most significant advances have occurred in the inter-relationships among cells, colonies and biofilm matrices. Indeed, the basis of one aspect of this invention is founded in the integration of the enlightened understanding of microorganism activity within the influence of the biofilm in which they reside.
Biofilms are matrix-enclosed accumulations of microorganisms such as bacteria (with their associated bacteriophages), fungi, protozoa and viruses that may be associated with these elements. While biofilms are rarely composed of a single cell type, there are common circumstances where a particular cellular type predominates. The non-cellular components are diverse and may include carbohydrates, both simple and complex, proteins, including polypeptides, lipids and lipid complexes of sugars and proteins (lipopolysaccharides and lipoproteins).
For the most part, the unifying theme of non-cellular components of biofilms is its backbone. In virtually all known biofilms, the backbone structure is carbohydrate or polysaccharide-based. The polysaccharide backbone of biofilms serves as the primary structural component to which cells and debris attach. As the biofilm grows, expands and ages along biologic and non-biologic surfaces in well-orchestrated enzymatic synthetic steps, cells (planktonic) and non-cellular materials attach and become incorporated into the biofilm. The growing biofilm not only attracts living cells; it also captures debris, food particles, cell fragments, insoluble macromolecules and other materials that add to the layer upon the polysaccharide backbone. In this fashion, layering continues and is repeated so that the initial layers i.e., those closest to the original polysaccharide backbone, become buried or embedded in the biofilm. As the biofilm ages, there are layers upon layers of polysaccharide backbone with the attendant cells, debris and insoluble macromolecular structures.
Biofilms are the most important primitive structure in nature. In a medical sense, biofilms are important because the majority of infections that occur in animals are biofilm-based. Infections from planktonic bacteria, for example, are only a minor cause of infectious disease. In industrial settings, biofilms inhibit flow-through of fluids in pipes, clog water and other fluid systems and serve as reservoirs for pathogenic bacteria and fungi. Industrial biofilms are an important cause of economic inefficiency in industrial processing systems.
Biofilms are prophetic indicators of life-sustaining systems in higher life forms. The nutrient-rich, highly hydrated biofilms are not just layers of plankontic cells on a surface; rather, the cells that are part of a biofilm are a highly integrated xe2x80x9ccommunityxe2x80x9d made up of colonies. The colonies, and the cells within them, express exchange of genetic material, distribute labor and have various levels of metabolic activity that benefits the biofilm as a whole.
Planktonic bacteria, which are metabolically active, are adsorbed onto a surface which has copious amounts of nutrients available for the initial colonization process. Once adsorbed onto a surface, the initial colonizing cells undergo phenotypic changes that alter many of their functional activities and metabolic paths. For example, at the time of adhesion, Pseudomonas aeruginosa (P. aeruginosa) shows upregulated algC, algD, algU etc. genes which control the production of phosphomanomutase and other pathway enzymes that are involved in alginate synthesis which is the exopolysaccharide that serves as the polysaccharide backbone for P. aeruginosa""s biofilm. As a consequence of this phenotypic transformation, as many as 30 percent of the intracellular proteins are different between planktonic and sessile cells of the same species.
In summary, planktonic cells adsorb onto a surface, experience phenotypic transformations and form colonies. Once the colonizing cells become established, they secrete exopolysaccharides that serves as the backbone for the growing biofilm. While the core or backbone of the biofilm is derived from the cells themselves, other components e.g., lipids, proteins etc, over time, become part of the biofilm. Thus a biofilm is heterogeneous in its total composition, homogenous with respect to its backbone and heterogeneous with respect it its depth, creating diffusion gradients for materials and molecules that attempt to penetrate the biofilm structure.
Biofilm-associated or sessile cells predominate over their planktonic counterparts. Not only are sessile cells physiologically different from planktonic members of the same species, there is phenotypic variation within the sessile subsets or colonies. This variation is related to the distance a particular member is from the surface onto which the biofilm is attached. The more deeply a cell is embedded within a biofilm i.e., the closer a cell is to the solid surface to which the biofilm is attached or the more shielded or protected a cell is by the bulk of the biofilm matrix, the more metabolically inactive the cells are. The consequences of this variation and gradient create a true collection of communities where there is a distribution of labor, creating an efficient system with diverse functional traits.
Biofilm structures cause the reduced response of bacteria to antibiotics and the bactericidal consequences of antimicrobial and sanitizing agents. Antibiotic resistance and persistent infections that are refractory to treatments are a major problem in bacteriological transmissions, resistance to eradication and ultimately pathogenesis. While the consequences of bacterial resistance and bacterial recalcitrance are the same, there are two different mechanisms that explain the two processes.
The use of enzymes in degrading biofilms is not new. Compositional patents as well as published scientific literature support the concept of using enzymes to degrade, remove and destroy biofilms. However, the lack of consistency in results and the inability to retain the enzymes at the site where their action is required has prohibited their widespread use.
As an alternative to enzymes, harsh chemicals, elevated temperatures and vigorous abrasion procedures are preferentially used over enzymes. There are conditions, however, where these non-enzymatic approaches cannot be used e.g., caustic- and acidic-sensitive environments, temperature or abrasion sensitive components that are associated with the biofilm and dynamic fluid action. When a biofilm is growing in an area where there is a constant fluid flow, the agents that remove biofilms are flushed away before they can carry our their desired function. This is particularly true for medical situations where aggressive sterilization procedures cannot be carried out and there is a desired fluid flow.
Removing and controlling biofilm growth in biologic media are specifically sensitive to harsh treatments. Biofilms in the oral cavity, on implanted devices and infections that occur in the alimentary and vaginal tracts or in eyes, ears etc. are particularly suited for an enzymatic treatment. There are also specific disease conditions, such as pneumonia and cystic fibrosis which are bacteria-based and occur in the lung, that would benefit from an enzymatic treatment only if the enzymes could be retained at the site long enough to fully realize their therapeutic actions.
Biofilm growth and the proliferation of infections in biologic systems are particularly sensitive to fluid-flow dynamics. Specific organs where infections occur e.g. eyes, oral cavity, gastrointestinal tract, vaginal tract, lungs etc., fluid and mucus flow is an integral part of the system""s normally functioning mode. Consequently, it is desirable to have the capability of removing unwanted biofilms in a non-harsh way in which the agent that acts on the biofilm is retained in close proximity to the biofilm and not swept away by fluids that are integral to the functioning system.
There are situations in or related to biologic systems where flow is minimal or non-existent. In these circumstances, the lack of demonstrated efficacy of enzymes to control biofilms is not related exclusively to their lack of ability to be retained at the site of the biofilm. Rather, the choice of enzyme to degrade the biofilm was inappropriate. An example is biofilm control on contact lenses and the cases or containers that hold the lenses when they are not in use. In these circumstances, it may not be a mandatory requirement for a means to retain the enzymes at or near the biofilm structure but only that the appropriate enzyme be part of the enclosed system.
It is also desirable to not only be able to degrade a biofilm within a biologic system, but also to be able to have a direct effect on the bacterial cells that are released as the biofilm is undergoing degradation. The combination of biofilm degradation and agents that directly affect bacterium is also not a new strategy. However, not infrequently in an open system, the same forces that flush or sweep away the biofilm degrading enzymes also flush bactericidal agents so that they cannot act directly upon bacteria unless there is a chance meeting between the agent and a planktonic bacterium.
Antibiotic/Antimicrobial Resistance. In the case of antibiotic or antimicrobial resistance, biofilms provide the unique opportunity for bacteria to reside in close proximity with one another for long periods of time. This prolonged juxtaposition of bacteria allows gene transfer between and among bacteria, allowing the genes of resistance to be transferred to same or different strains of bacteria to neighboring cells that are not resistant. Consequently, a virulent cell can transfer its virulence genes to a non-virulent cell, making it resistant to antibiotics.
Antibiotic/Antimicrobial Recalcitrance. In the case of antibiotic or antimicrobial recalcitrance, there are two possible explanations, both of which involve the biofilm and both of which may be operative simultaneously. While gene transfer may occur, it is not a factor in recalcitrance.
The first of the explanatory mechanisms is simply a physical phenomenon: the biofilm structures present a barrier to penetration of antibiotics and antimicrobial agents and a protective shroud to physical agents such as ultraviolet radiation. The biofilm, with its polysaccharide backbone and residual debris that is associated with the biofilm, provides a barrier to deep-seated bacteria. Unless the biofilm is removed or disrupted, complete cellular kill within the biofilm structure is not achieved by chemical or physical agents.
The second explanatory mechanism is based on biochemical or metabolic principles. Just as the deep-seated bacteria are protected from chemical and physical agents by the xe2x80x9cbarrierxe2x80x9d effect of the biofilm, the biofilm also acts as a barrier to nutrients that are necessary for normal metabolic activity. Further, the nutrient-limited bacteria are in a reduced state of metabolic activity, which make them less susceptible to chemical and physical agents because the maximal effects of these killing agents are achieved only when the bacteria are in a metabolically active state.
With any of the possible mechanistic explanations for either resistance or recalcitrance, removal or disruption of the biofilm is a mandatory requirement. Stripping away of the biofilm components e.g., the polysaccharide backbone with the accumulated debris accomplishes several objectives: 1) reduced opportunity for gene transfer; 2) increased penetration of chemical and physical agents; and 3) increased free-flow of nutrients which would elevate the metabolic activity of the cells and make them more susceptible to chemical and physical agents. Furthermore, removal or disruption of the biofilm will free cells from a sessile state to make them planktonic which also increases their susceptibility to chemical and physical agents.
Prevention of Biofilm Formation. Under ideal conditions for controlling biofilms, the preferred approach for limiting the detrimental effects of biofilms is prevention of initial colonization by cells. For the most part, these approaches focus on the environment in which planktonic bacteria are present without particular attention to the bacteria themselves. This can be done to a limited extent through physical means e.g., electrical charges etc., chemical strategies e.g., surface coatings (paints and varnishes with antimicrobial chemicals) etc. and biochemical means e.g. nutrient limitation. However, for the majority of situations when fouling by biofilms occurs, these strategies are not practical or at best have limited utility.
Limiting Early Biofilm Growth. The next line of defense against the adverse effects of biofilms revolves around curtailing the consequences of the post-initial colonization of planktonic bacteria to a surface by limiting the initial proliferation of the biofilm. This can be accomplished, only to a limited extent, by continual disruption of early, immature biofilms or by inhibiting the biosynthesis of the structural exopolysaccharide backbone. Interdiction of early exopolysaccharide synthesis is usually achieved by macrolide antibiotics e.g., large ring lactones, erythromycin being one example. This later course of action constitutes a shift from an attempt to control the biofilm structure or environment to a direct action upon the living cells within the biofilm.
Destroying Established Biofilms. For established biofilms, with various levels of embedded cells, disruption, fragmentation and removal of the biofilm is necessary. This can be accomplished, under limited circumstances, with physical means e.g., abrasion methods, sonication, electrical charge stimulation, detergent and enzymatic. There are obvious drawbacks to any one method, precluding a universal method or approach. However, the common trait of all of these methods lies in their focus on the biofilm structure and not the living cells within the biofilm.
If, by any one of the methods, the structure of the biofilm is altered or disturbed, a secondary, complementary attack on the living cells within the biofilm can be made with antibiotics and antimicrobial agents.
An important aspect of the invention lies in two concepts, both of which may operate independently, but when combined, they effectively remove biofilms and prevent their reestablishment. The first of these is the removal of the biofilm structure in an orderly and controlled manner. The second concept is a specific consequence of removing the biofilm structure. During the removal or dismantling of the biofilm structure, especially the exopolysaccharide backbone, cells within the biofilm become more susceptible to the bactericidal action of antimicrobials, antibiotics, sanitizing agents and host immune responses. As the biofilm is removed, some cells within the biofilm are liberated and become planktonic; others, however, remain sessile but are more vulnerable to being killed because the protective quality of the biofilm is reduced.
One aspect of the invention consists of one or more hydrolytic enzyme(s) whose specificity includes its (their) ability to degrade exopolysaccharide backbone structure(s) of a biofilm produced by bacterial strain(s). Attached to the enzyme(s), either through chemical synthetic procedures or recombinant technology, are one or more moieties that have the capability of binding, either reversibly, in a non-covalently, or irreversibly (covalent bonded) to a surface near the biofilm or the biofilm itself. This aspect is directed at the degradation of the biofilm backbone structure.
Another aspect of the invention consists of two or more hydrolytic enzymes. One enzyme has the specificity to degrade the biofilm""s exopolysaccharide backbone structure of a biofilm; at least one other enzyme is hydrolytic in nature, having the capability to degrade proteins, polypeptides, lipids, lipid complexes of sugars and proteins (lipopolysaccharides and lipoproteins). Attached to the enzymes, either individually or collectively as a single unit through chemical synthetic procedures or recombinant technology, are one or more moieties that have the capability of binding either reversibly, non-covalently, or irreversibly (covalent bonded) to a surface near the biofilm or the biofilm itself. This aspect is directed at the degradation and removal of the biofilm backbone structure along with any other materials that may be associated with the backbone, which collectively constitute the entire biofilm.
Still another aspect of the invention consists of two or more enzymes, wherein at least one enzyme has the capability of degrading a biofilm structure produced by a bacterial strain, or a mixed combination of various strains, and the other enzymes(s) has (have) the capability of acting directly upon the bacteria, causing lysis of the bacterial cell wall. One or more moieties are attached to the enzymes, forming either a single unit or multiple units. The moieties are attached to the enzymes either through chemical synthetic procedures or recombinant technology to give the enzyme moiety the capability of binding either reversibly, non-covalently, or irreversibly (covalent bonded) to a surface near the biofilm or the biofilm itself. The purpose of this multi-enzyme system is directed at the degradation and removal of the biofilm with the contemporaneous bactericidal consequences for bacteria that were embedded in the biofilm""s structure.
A fourth aspect of the invention consists of two sets of enzymes, the first being one or more enzymes with the appropriate anchor attached to the enzyme(s) for the purpose of degrading the biofilm structure; the second set of enzymes are also connected to anchor molecules whose function is to generate active oxygen to directly attack and kill bacteria that are exposed during the process of the degradation and removal of the biofilm.
A fifth aspect of the invention consists of one or more enzyme complexes to degrade biofilm structures and a second component of one or more unbound or free non-enzymatic bactericidal components whose function is to kill newly exposed bacteria as the biofilm structure is removed. The non-enzymatic bactericidal agents include, but are not limited to, antimicrobial agents, antibiotics, sanitizing agents and host immune response elements.
The purpose of these various embodiments is to hold or retain the biofilm-degrading enzymes and bactericidal components in fluid-flow systems that are open, partially open or, at least not completely closed systems. Without the capability to keep the appropriate active agents at or near the biofilm structure, they may be swept away in the fluid flow.
The above five previously described aspects of the invention apply to open or partially open systems where there is fluid flow. However, there is also an additional embodiment for completely closed systems in which the enzyme or antibacterial agent may or may not have a binding moiety attached to.
A sixth aspect of the invention consists of one or more appropriately selected enzymes, not being connected to a binding moiety but limited by their ability to degrade a biofilm that is contained within such a closed system where there is minimal to no fluid flow.